1. Field of the Invention
The present invention relates to the production of novel synthetic peptide (polypeptide) sequences based upon information derived from natural peptide sequences and the use of these peptide sequences as antigens in the production of vaccines, antiviral agents, diagnostic reagents and the like; for the treatment of infectious and immune diseases, such as cancer, hoof and mouth disease and the like in mammals, specifically cattle and human beings. The process includes the prediction of the antigenic determinant site (.beta.-turn) in a protein; chemically synthesizing the peptide sequences; optionally binding the peptide to a high molecular weight carrier, such as a protein (BSA, thyroglobulin, KLH bovine gamma globulin and the like); introducing the peptide into a host as the peptide or optionally its conjugate; and immunologically producing antibodies to the peptide.
2. State of the Art
The development of synthetic peptides useful in biological applications, such as synthetic vaccines, has been under investigation for many years. The selection of and chemical or biological production of specific peptide sequences has received particular attention.
Protein structure can be visualized as a hydrophobic core of amino acids surrounded by a shell of more polar amino acids which are accessible to the solvent at the surface of the molecule. In protein molecules which interact with a receptor, such as protein hormones, the interaction between the protein and the receptor must take place at the surface-accessible sites while the hydrophobic core provides the three dimensional stability of the molecule. By arranging the critical binding site residues in the appropriate conformation, it is possible to synthesize small fragments of the protein molecule which mimic the essential surface features of the present protein and retain the appropriate biological activity. The same criteria for choosing possible binding regions of protein molecules, the most important being surface exposure and appropriate conformation can also be used to predict antigen binding sites.
Chou and Fasman, for example, reported in Biochemistry, vol. 13, pp 212-245 in 1974 that protein structures determined by x-ray crystallography were analyzed, and they then calculated the probabilities that each of the normal amino acid residues would be in an .beta.-helix, .beta.-pleated sheet or a .beta.-turn type of structure. From these probability coefficients, they developed a method for predicting protein structure. For instance, the Chou-Fasman method for predicting the secondary structure (including .alpha.-helix and .beta.-pleated sheet residues) for fibroblast (F) and leukocyte (Le) interferons from the amino acid sequences is described by T. Hayes in Biochem. and Biophys. Res. Comm., Vol. 95, No. 2, pp 872-9, published in 1980.
Several other methods have been applied to the prediction of the secondary structure of proteins. For example, the methods of Burgess/Sheraga (Israel J. Chem., Vol. 12, pp 239-286, published in 1974, and V. I. Lim (J. Mol. Biol., vol. 88, pp 857-894, published in 1974) have been used in a similar manner.
A computer program (SOAP) that progessively evaluates the hydrophilicity and hydrophobicity of a protein along its amino acid sequence is described by J. Kyte and R. F. Doolittle, J. Mol. Biol., Vol. 157, pp 105-132 (1982), which is incorporated herein by reference.
T. Hopp has reported that the most hydrophilic portion of a protein molecule will be the most exposed on the surface of a protein and will constitute an antigenic determinant. Only the most hydrophilic region was reported to be an antigenic determinant (Immuno., Vol. 76, No. 6, pp 3824-28, published June 1981). Hence, this predictive technique is said to yield only one antigenic determinant on the molecule. In a more recent publication, Hopp retracted this claim, see Genetic Engineering News, vol. 1, p 1 (1981).
Another group has reported that the same approach, i.e. calculation of regions of minimum hydrophobicity (therefore, maximum hydrophilicity) using essentially the program of Kyte and Doolittle, predicts antigenic determinants. (See, for example, R. A. Lerner, et al., Cell., Vol. 23, pp 309-310, published in 1981). In practice the latter group synthesized overlapping fragments constituting the entire length of the protein chain.
The preparation of antigenic hapten-carrier conjugates has been discussed by B. F. Erlanger in Methods in Enzymology, Vol. 70, pp. 85-104, published in 1980 by Academic Press, Inc. of New York, N.Y.; and the structure and specificity of synthetic polypeptide antigens is discussed by M. Sela in the Ann. N.Y. Acad. Sci., Vol. 169, pp 23-35 (1970).
The problems associated with the development of synthetic polypeptide vaccines is discussed by A. J. Zuckerman in Nature, Vol. 295, pp 98-9, published Jan. 14, 1982; and by N. Wade in Science, Vol. 213, pp 623-8, published Aug. 7, 1981.
G. R. Dreesman et al., in Nature, vol. 295, pp 158-160, published on Jan. 14, 1982, discusses the selection and preparation of synthetic polypeptides which elicit an antibody response for hepatitis B surface antigen in mice after a single injection. The amino acid sequences described are different from the sequences of a natural protein.
Antibodies specific for the amino and carboxyl-terminal portions of simian virus 40 large T antigen are obtained by the immunization of rabbits with synthetic peptides corresponsing to these regions. The procedures used in the preparation of antibodies specific for the ends of large T antigen with synthetic peptides as antigens are discussed by G. Walter et al., Proc. Natl. Acad. Sci. USA, Vol. 77, No. 9, pp 5197-5200, published in September, 1980.
The synthesis of antigenically active polypeptides and a process for their manufacture are described in U.S. Pat. Nos. 4,327,075 and 4,193,915, which are incorporated herein by reference. Additional related information is also found in the foreign patent literature; e.g., EPO Patent Nos. 13 828 and EPO 44 710, which are incorporated herein by reference.
This invention will provide a cheaper, more efficient method of producing biologically active polypeptides or their conjugates which are useful in the diagnosis and treatment of diseases in mammals. Further, the synthetic vaccines containing polypeptides described herein would be purer and safer than conventional vaccines, which presently consist of the killed or attenuated whole virus and sometimes debris from the culture medium as well. Such synthetic vaccines will also be safer to manufacture since the risk of contamination of the site with pathogens will not be present.